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Results for “Codon, Terminator”

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PubMed1999

[The proline biosynthesis gene proA from the thermophilic bacterium Thermus ruber: its cloning, sequencing and heterologous expression].

The proA proline biosynthesis gene of thermophilic bacterium Thermus ruber was cloned and sequenced, and several properties of the encoded enzyme, gamma-glutamylphosphate reductase (GPR) were studied. The proA open reading frame (ORF) was of 1286 bp. Nucleotide sequence analysis revealed the ATG initiation codon in position 36 and the TTA termination codon in position 1304. A deduced protein product of the gene was shown to be of 44,919 Da in molecular weight. The GC content was 66%, as is characteristic of various bacteria of the genus Thermus. An amino acid sequence encoded by the cloned gene showed the highest homology (up to 64%) with GPR of T. thermophilus. The maximum activity of GPR (8.2 x 10(-2) units/ml) was observed at 55 degrees C. A weak enzymatic activity was also detected at 70 degrees C. The enzyme can be used in biotechnological studies.

Aldehyde Oxidoreductases↗

PubMed1990

[Molecular diagnosis of inherited coagulation disorders--sequence analysis of hemophilia B patients with anti-factor IX antibodies].

Seven hemophilia B patients with anti-factor IX antibodies are studied with molecular means. A total factor IX gene deletion was detected in four patients from three distinct families. Three other patients without detectable gene arrangements were investigated at the DNA sequence level. Enzymatic amplification of the factor IX gene and subsequent DNA sequencing revealed four novel nucleotide mutations in these patients. Patient HB 5 had two point mutations in his factor IX gene. One is located at nucleotide -793 from the translation start (G-A) and the other (C-T) was found in the codon for 191-Gln changing to a termination codon. Patient HB6 had a point mutation (G-A) in the splicing junction of intron g. A2 bp nucleotide deletion was detected in the third exon of the patient HB7 yielding 8 frameshifted amino acids and a stop codon. These results suggested that not only a large deletion of the factor IX gene but also point mutations or small deletion of the gene which may cause the substantial loss of the coding information for the mature protein are involved in the development of anti-factor IX antibodies in hemophilia B patients.

Antibodies↗

PubMed1980

Structural comparison of two nontandemly repeated yeast glyceraldehyde-3-phosphate dehydrogenase genes.

A hybrid plasmid (pgap63) was isolated which contains a second yeast glyceraldehyde-3-phosphate dehydrogenase structural gene. The complete nucleotide sequence of this gene was determined and compared with the primary structure of a yeast glyceraldehyde-3-phosphate dehydrogenase gene (pgap49) which was reported previously (Holland, J.P., and Holland, M.J. (1979) J. Biol. Chem. 254, 9839-9845). Based on the restriction endonuclease cleavage maps of the isolated segments of yeast DNA which contain these genes, the two genes are nontandemly duplicated. Greater than 94% of the nucleotides within the coding regions of these genes are homologous and the polypeptides encoded by the two structural genes differ by only 15 amino acid residues. Both genes have the same, highly biased, codon usage pattern and neither contains intervening sequences. Approximately 100 nucleotides adjacent to the ATG initiation codons and 130 nucleotides beyond the TAA termination codons are greater than 70% homologous. Structures within the flanking sequences of the genes which are potentially relevant to transcriptional and translational control are described. Several sequences (8 to 15 nucleotides in length) are repeated in both the 5' and 3' flanking sequences of the genes in a noninverted fashion. Finally, a rapid procedure for the isolation of spontaneous deletions within hybrid plasmid DNAs is described, as is the isolation of a structural gene deletion in pgap49.

Amino Acid Sequence↗

PubMed1995

Molecular basis for a familial defect in phagocyte expression of IgG receptor I (CD64).

Four individuals have been identified, within a single family, who lack phagocyte expression of the high affinity type I IgG receptor (CD64). As a result, their monocytes are unable to support mouse IgG2a anti-CD3-induced T cell mitogenesis (nonresponder individuals). Southern blotting proved all three human Fc gamma receptor I (hFc gamma RI) genes to be present in nonresponders without major structural changes. Nucleotide sequencing showed identical hFc gamma RIA promoter regions in all individuals. At the message level, a distinct difference was noted between monocytes from control (responder) donors and from nonresponders. Both a 1.7- and 1.6-kb message were found in responders, whereas in nonresponders only the 1.6-kb species was detectable. Reverse transcriptase-PCR analyses showed the hFc gamma RIa transcript (encoding a receptor with three extracellular Ig-like domains) to be present at a approximately 15- to 20-fold lower level in nonresponder monocytes. Importantly, we found a single nucleotide difference (C --> T) within the extracellular domain exon 1-encoding region of hFc gamma RIA in nonresponders, resulting in the change of codon 92 (encoding an arginine) into a termination codon. This change likely affects mRNA stability and, thereby, leads to undetectable expression of phagocyte-hFc gamma RIa. Despite this defect, these individuals are apparently healthy, suggesting that hFc gamma RIa is dispensable for phagocyte functioning.

Adult↗

PubMed2000

Familial British dementia: expression and metabolism of BRI.

Vidal et al. (1999. Nature 399: 776-778) discovered that the underlying genetic lesion in familial British dementia (FBD) is a T-A transversion at the termination codon of a membrane protein, termed BRI. The mutation creates an arginine codon; translational read-through generates a novel protein, termed BRI-L, that is extended by 11 amino acids at the carboxyl-terminus. BRI-L is the precursor of the ABri peptide, a component of amyloid deposits in FBD brain. We demonstrate that both BRI and its mutant counterpart are constitutively processed by furin, resulting in the secretion of carboxyl-terminal peptide derivatives that correspond to all, or part of, ABri. Notably, elevated levels of peptides are generated from the mutant BRI precursor, suggesting that subtle conformational alterations at the carboxyl-terminus may influence furin-mediated processing. We have examined BRI/BRI-L processing by other members of the prohormone convertase (PC) family (PACE4, LPC, PC 5/6) and found that these enzymes also process BRI, albeit inefficiently. Moreover, BRI-L processing by the other PC members is severely compromised. Finally, our electron microscopic studies reveal that synthetic ABri peptides assemble into insoluble beta-pleated fibrils. Collectively, our results support the view that enhanced furin-mediated processing of mutant BRI generates amyloidogenic peptides that initiate the pathogenesis of FBD.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Regulated translation termination at the upstream open reading frame in s-adenosylmethionine decarboxylase mRNA.

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a cis-acting element that confers feedback control by cellular polyamines on translation of this message. Recent studies demonstrated that elevated polyamines inhibit synthesis of the peptide encoded by the uORF by stabilizing a ribosome paused in the vicinity of the termination codon. These studies suggested that polyamines act at the termination step of uORF translation. In this paper, we demonstrate that elevated polyamines stabilize an intermediate in the termination process, the complete nascent peptide linked to the tRNA that decodes the final codon. The peptidyl-tRNA molecule is found associated with the ribosome fraction, and decay of this molecule correlated with release of the paused ribosome from the message. Furthermore, the stability of this complex is influenced by the same parameters that influence regulation by the uORF in vivo, namely the concentration of polyamines and the sequence of the uORF-encoded peptide. These results suggest that the regulated step in uORF translation is after formation of the peptidyl-tRNA molecule but before hydrolysis of the peptidyl-tRNA bond. This regulation may involve an interaction between the peptide, polyamines, and a target in the translational apparatus.

Adenosylmethionine Decarboxylase↗

PubMed1977

Hemoglobin and the genetic code. Evolution of protection against somatic mutation.

One-half of the twenty amino acids of the genetic code are just one mutational step away from the chain-terminator codons UAA, UAG, and UGA. It is postulated that somatic mutation to terminator is a hazard to which the organism has and to respond by adjusting certain proteins in the direction of fewer mutable residues. This view is supported by calculations based on the primary structure of five of the human hemoglobin chains. Each chain is scored for mutability to terminator in accord with the numbers and kinds of amino acids present. Among the adult chains, the most essential one, the alpha, has lowest mutability. The beta and delta follow, and in order of the presumed harm to the organism of a shortage of chain copies. Ante-natal chains tend to have higher mutabilities, supporting the view that cumulative mutational change in DNA can do little if the gene ceases to transcribe early in life. Two other predicitons based on the supposition of effective selection against mutability to terminator are also met: chain length of polypeptides is negatively correlated with their scores for mutability to terminator, and examination of the recently determined sequence of beta messenger RNA shows preferential use of codons that are not readily mutable to terminator.

Amino Acid Sequence↗

PubMed1990

Frameshifting at the internal stop codon within the mRNA for bacterial release factor-2 on eukaryotic ribosomes.

A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence. High-efficiency frameshifting around this codon can occur on eukaryotic ribosomes as well as prokaryotic ribosomes. This was determined from the relative efficiency of translation of RF-2 RNA compared with that for the other release factor RF-1, which lacks the in-frame premature stop codon. Since the termination product is unstable an absolute measure of the efficiency of frameshifting has not been possible. A gene fusion between trpE and RF-2 was carried out to give a stable termination product as well as the frameshift product, thereby allowing a direct determination of frameshifting efficiency. The extension of RF-2 RNA near its start codon with a fragment of the trpE gene, while still allowing high efficiency frameshifting on prokaryotic ribosomes, surprisingly gives a different estimate of frameshifting on the eukaryotic ribosomes than that obtained with RF-2 RNA alone. This paradox may be explained by long distance context effects on translation rates in the frameshift region created by the trpE sequences in the gene fusion, and may reflect that pausing and translation rate are fundamental factors in determining the efficiency of frameshifting.

Base Sequence↗

PubMed1998

The hypotrichous ciliate Euplotes octocarinatus has only one type of tRNACys with GCA anticodon encoded on a single macronuclear DNA molecule.

Deviations from the universal genetic code have evolved independently several times in ciliated protozoa. Thus, in some species UAA and UAG are no longer used as termination codons, but are read as glutamine, whereas in the genus Euplotes , UGA is translated as cysteine. We have investigated the nature of the tRNACys isoacceptor responsible for decoding UGA in Euplotes cells. Southern hybridization analyses indicated that a single DNA molecule of 630 bp encoding tRNACys exists in the macronucleus of Euplotes octocarinatus . Cloning and sequencing of this fragment revealed that it contains only one copy of a tRNACys gene, which codes for a normal tRNACys with GCA anticodon. This is the first report of the characterization of a tRNA gene in any hypotrichous ciliate. It contains putative signals for initiation and termination of transcription by RNA polymerase III and can be transcribed efficiently in vitro in HeLa cell nuclear extract. Intensive studies on the DNA and tRNA level involving PCR analyses have not disclosed the existence of any tRNA Cys isoacceptor with UCA or ICA anticodons. Translation of the UGA codon by tRNA sub GCA sup Cys necessitates a G:A mispairing in the first anticodon position. We discuss a number of aspects which might contribute to the finding that a near-cognate tRNA isoacceptor efficiently translates the UGA stop codon.

Animals↗

PubMed2005

Characterisation of the mulibrey nanism-associated TRIM37 gene: transcription initiation, promoter region and alternative splicing.

The TRIM37 gene encodes a peroxisomal protein of unknown function. Mutations in TRIM37 underlie mulibrey nanism, a rare autosomal recessively inherited disorder with severe growth failure of prenatal onset, constrictive pericardium, hepatomegaly and characteristic dysmorphic features. Eleven mulibrey nanism-associated mutations have been identified. We here characterised TRIM37 further by mapping the transcription initiation site and promoter region as well as by analysing splice variants. By primer extension analysis, several transcription initiation sites were localised to a region between -246 and -373 relative to the ATG codon for translation initiation. Basal promoter activity was mapped within 600 nucleotides upstream from the translation initiation site using promoter-luciferase reporter constructs. Several alternative splice variants of TRIM37 exist in databases. Most of these predict non-functional protein products, are expressed at low levels and are thus likely to be targets for nonsense-mediated mRNA decay. A novel splice variant, TRIM37b, with an alternative termination codon and 3'untranslated region (UTR) transcribed from an exon 16 kb downstream from exon 24, predicts an identical protein product with the previously identified transcript, TRIM37a. As seen by Northern blot analysis and quantitative real-time PCR, both transcripts are highly expressed in testis, whereas in other tissues TRIM37a is prominent. The 3'UTR of the PPM1E gene in the opposite strand overlaps TRIM37b. These data suggest that TRIM37 expression is regulated by several mechanisms: through nonsense surveillance of non-functional transcripts, as well as through 3'UTR regulatory sequences and/or naturally occurring antisense RNAs especially in testis.

3' Untranslated Regions↗

PubMed2001

A dysfunctional factor X (factor X San Giovanni Rotondo) present at homozygous and double heterozygous level: identification of a novel microdeletion (delC556) and missense mutation (Lys(408)-->Asn) in the factor X gene. A study of an Italian family.

Low levels of factor X (F.X) were detected in a 4-year-old boy who experienced acute lymphoblastic leukemia and bleeding manifestations. Laboratory data suggested the presence of a dysfunctional F.X molecule. Two novel F.X gene mutations were identified in the proband that was double heterozygous for both: a microdeletion (delC556) in exon VI resulting in a frameshift leading to a termination codon at position 226. This deletion was found in six family members with reduced F.X antigen and activity levels. A second mutation characterised by a G(1344)-->C transversion in exon VIII was detected in the proband resulting in a Lys(408)-->Asn substitution. This latter mutation was present in several asymptomatic family members from the paternal and the maternal side. The proband's sister was homozygous for the Lys(408)-->Asn substitution and exhibited low F.X activity with a normal antigen level. The naturally occurring F.X Lys(408)-->Asn (F.X(K408N)) variant was isolated from plasma of either homozygous or double heterozygous individuals. NH(2)-terminal sequencing of the heavy chain of F.X(K408N) failed to show any sequence abnormality in patients who were also carriers of the delC556, suggesting that this latter lesion accounted for the lack of F.X synthesis. Purified F.X Lys(408)-->Asn had an identical behaviour to normal F.X as judged by SDS-PAGE and immunoblotting. Clotting assay using purified F.X(K408N) and F.X-deficient plasma resulted in a laboratory phenotype similar to that observed in a homozygous subject for F.X Lys(408)-->Asn substitution. This is the first characterisation of a naturally occurring F.X variant with a mutation at the COOH-terminal end of the molecule.

Amino Acid Sequence↗

PubMed1997

ITIH1*Q0iwate, a null allele of inter-alpha-trypsin inhibitor H1 caused by deletion/frameshift mutation.

The molecular characterization of the first example of null allele in the inter-alpha-trypsin inhibitor H1 (ITIH1) system, ITIH1*Q0iwate, encountered as apparent inverse homozygosity of ITIH1 phenotypes between mother and child in a paternity case, is described. Single-strand conformation polymorphism analysis and subsequent sequencing showed that deletion of a single nucleotide in the codon for Lys87 results in a frameshift causing a terminator codon downstream of the deletion. This leads to premature termination of ITIH1 protein translation at amino acid 128, resulting in a truncated protein.

Alleles↗

PubMed1993

Lack of peptide-release activity responding to codon UGA in Mycoplasma capricolum.

In Mycoplasma capricolum, a relative of Gram-positive eubacteria with a high genomic AT-content (75%), codon UGA is assigned to tryptophan instead of termination signal. Thus, in this bacterium the release factor 2 (RF-2), that recognizes UAA and UGA termination codons in eubacteria such as Escherichia coli and Bacillus subtilis, would be either specific to UAA or deleted. To test this, we have constructed a cell-free translation system using synthetic mRNA including codon UAA [mRNA(UAA)], UAG [mRNA(UAG)] and UGA [mRNA(UGA)] in-frame. In the absence of tryptophan, the translation of mRNA(UGA) ceased at UGA sites without appreciable release of the synthesized peptides from the ribosomes, whereas with mRNA(UAA) or mRNA(UAG) the bulk of the peptides was released. Upon addition of the E.coli S-100 fraction or B.subtilis S-100 fraction to the translation system, the synthesized peptides with mRNA(UGA) were almost completely released from the ribosomes, presumably because of the presence of RF-2 active to UGA in the added S-100 fraction. These data suggest that RF-2 is deleted or its activity to UGA is strongly weakened in M.capricolum.

Base Sequence↗

PubMed1998

Novel mutation of the DAX1 gene in a patient with X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism.

X-linked adrenal hypoplasia congenita (AHC) is characterized by primary adrenal insufficiency and is frequently associated with hypogonadotropic hypogonadism (HHG). Mutations of the DAX1 gene have been reported in patients with AHC and HHG. We found a novel DAX1 mutation in our patient. Sequence analysis of the patient's DAX1 demonstrated a 1-bp (G) deletion at codon 49 in exon 1. The mutation shifts the reading frame, resulting in completely different amino acid sequences from codon 49 to the premature stop codon at 84. The G was present at this position in the sequences of the father and 2 younger brothers. Direct sequence and single-strand conformation polymorphism analyses of polymerase chain reaction fragments revealed that the mutation at codon 49 was heterozygously present in the mother's DAX1 gene. The codon 84 is located in the first half of the DNA binding domain, and the mutation site is closer to the N-terminus than those in previously reported cases. The onset of adrenal insufficiency in the neonatal period as seen in our patient has also been reported in other patients with different DAX1 mutations, especially in a patient with DAX1 protein lacking 11 amino acids at the C-terminus. Therefore, it is less likely that position of termination codons correlate to clinical manifestations.

Adrenal Glands↗

PubMed2000

Three different premature stop codons lead to skipping of exon 7 in neurofibromatosis type I patients.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder affecting one in 3,500 individuals. The mutation rate in the NF1 gene is one of the highest known for human genes. Compared to other methods, the protein truncation test (PTT) provides improved efficiency in detecting NF1 mutations which are dispersed throughout the gene which spans 350 kilobases of genomic DNA. We have applied the PTT and subsequent sequence analysis of cloned cDNA to identify mutations in NF1 patients. We report here the identification of two novel (W336X and Q315X), and one recurrent (R304X) mutation located in exon 7 and show that all three premature termination codons lead to skipping of exon 7 in a proportion of the transcripts derived from the mutated allele. Possible mutation-induced alterations of the RNA secondary structure and their impact on skipping of exon 7 of the NF1 gene are explored and discussed.

Base Sequence↗

PubMed1984

Isolation and sequence of the structural gene for cytochrome c oxidase subunit VI from Saccharomyces cerevisiae.

Using synthetic oligodeoxyribonucleic acid probes we have identified and isolated COX6, the structural gene for subunit VI of cytochrome c oxidase from Saccharomyces cerevisiae. The nucleotide sequence of COX6 predicts an amino acid sequence, for the mature subunit VI polypeptide, which is in perfect agreement with that determined previously. The nucleotide sequence of COX6 also predicts that subunit VI is derived from a precursor with a highly basic 40-amino acid NH2-terminal presequence. This precursor has been observed after in vitro translations programmed by yeast poly(A+)RNA. Northern blot analysis of poly(A+) RNA from strain D273-10B reveals that COX6 is homologous to three RNAs of 1800, 900, and 700 bases in length. By means of Southern blot analysis, the cloned gene was shown to be co-linear with yeast chromosomal DNA and to exist in a single copy in the yeast genome. An additional open reading frame, consisting of 82 codons, terminates 22 codons upstream from COX6. It is "in frame" with the COX6 coding region.

Amino Acid Sequence↗

PubMed2003

Nonsense mediated decay downregulates conserved alternatively spliced ABCC4 transcripts bearing nonsense codons.

Drug transporters are an important part of the defense of cells against cytotoxic agents. One major group of transporters is known as multidrug resistance associated proteins (MRP; ABCC gene family). The MRPs belong to the ATP binding cassette transporter superfamily. One family member, ABCC4 (also known as MRP4) functions as a cellular efflux pump for anti-HIV drugs, such as 9-(2-phoshoenylmethoxyethyl) adenine and azido-thymidine-monophosphate, an antiviral nucleotide, ganciclovir-monophosphate, and anti-cancer agents such as thiopurines. We isolated a ABCC4 cDNA encoding a non-functional protein, owing to an insertion, and subsequently determined the ABCC4 gene structure. This analysis revealed that the insertion was attributed to two additional exons that would be predicted to produce premature termination codons (PTC) in ABCC4. The highly similar mouse Abcc4 gene also contained these exons, which were remarkable because their size and sequence identity were much higher than the overall similarity between these genes. Further, a comparison of human, monkey and rodent ABCC4 genes revealed that these same PTC-producing exons were also highly conserved in evolution. As all the ABCC4 mRNA containing these PTC exons might produce nonsense mRNA, we further tested the hypothesis that these mRNAs were targets of nonsense-mediated mRNA decay (NMD). Protein synthesis inhibition selectively stabilized PTC containing ABCC4 transcripts in human, monkey and rodent cell lines. Moreover, the amount of PTC-containing ABCC4 transcripts was critically dependent upon protein synthesis, as removal of the inhibitor dramatically decreased expression, which correlated with the resumption of protein synthesis. These are the first studies to indicate that the highly conserved PTC exons of the ABCC4 gene may dictate its expression.

Alternative Splicing↗

PubMed1996

A splicing-dependent regulatory mechanism that detects translation signals.

Premature termination codons (PTCs) can cause the decay of mRNAs in the nuclear fraction of mammalian cells. This enigmatic nuclear response is of interest because it suggests that translation signals do not restrict their effect to the cytoplasm, where fully assembled ribosomes reside. Here we examined the molecular mechanism for this putative nuclear response by using the T-cell receptor-beta (TCR-beta) gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic ontogeny. We found that PTCs had little or no measurable effect on TCR-beta pre-mRNA levels, but they sharply depressed TCR-beta mature mRNA levels in the nuclear fraction of stably transfected cells. A PTC split by an intron was able to trigger the down-regulatory response, implying that PTC recognition occurs after an mRNA is at least partially spliced. However, intron deletion and addition studies demonstrated that a PTC must be followed by at least one functional (spliceable) intron to depress mRNA levels. One explanation for this downstream intron-dependence is that cytoplasmic ribosomes adjacent to nuclear pores scan mRNAs still undergoing splicing as they emerge from the nucleus. We found this explanation to be unlikely because PTCs only 8 or 10 nt upstream of a terminal intron down-regulated mRNA levels, even though this distance is too short to permit PTC recognition in the cytoplasm prior to the splicing of the downstream intron in the nucleus. Collectively, the results suggest that nonsense codon recognition may occur in the nucleus.

Animals↗